Streptomycyl amines and production thereof



Patented Aug. 19, 1952 STREPTOMYCYL AMINES AND PRODUCTION THEREOF Walter A. Winsten, Forest Hills, Murray Katz, New York, and Sidney R. Safir, White Plains, N. Y., assignors to Schenley Industries, Inc., a

corporation of Delaware No Drawing. Application August 15 1947,

I Serial No. 768,940

7 Claims.

Our invention relates to antibiotic derivatives of streptomycin and method of makingth same.

Streptomycin is an antibiotic which is obtained from certain strains of Strcptomyces grzseus. It is well known that streptomycin is substantially wholly antibiotically inactivated by reacting it with certain compounds which have alphaamino-beta mercapto groups. Among these compounds which substantially inactivate streptomycin, are l-cysteine, cysteinyl-glycine, dlbeta beta dimethylcysteine, l-cysteine ethyl ester, and so forth.

The compound above designated as l-cysteine is also designated as 'l-2-amino-3-mercaptopropanoic acid, and also as l-beta-mercaptoalanine. Its formula is HS.OH2.GII-(NH2) 0001-11 The formula of lalanine, also designated as l-Z-aminopropanoic acid, is

As later mentioned, we use cysteine hydrochloride in order to produce the inactive derivative of streptomycin. The formula of cysteine hydrochloride is We have discovered that if the resultant substantially inactive streptomycin derivatives are treated with Raney nickel so as to produce simultaneous desulfurizing action and reducing action, we produce new derivatives of streptomycin which are active as antibiotics against a-lar number of gram-positive and gram-negative rganisms.

When reference is made herein to alpha amino mercapto compounds, we designate organic compounds in which the amino group, and the sulphydryl or mercapto group, are on adjacent carbon atoms of a straight carbon chain.

These new derivatives are not inactivated by said alpha amino mercapto compounds, and by many other compounds which inactivate streptomycin; I I

Streptomycin has a free aldehyde-carbonyl group which is apart of the streptose' portion of the molecule. The composition 'of streptomycin is set forth by Kuehl et al., in Journal of American Chemical 'Society (1946), vol. 68, Page According to Kuehl et al., streptomycin con sists of a diguanid-ine base which is designated as streptidine, linked glucosidically w a disa'ccharide moiety designated as streptobiosamine Streptobiosamine consists of streptose, linked glucosidioally to N-methyl-l-glucosamine.

For convenience, hereinafter the term Strep designates the streptomycin molecule, with the exception of the streptose aldehyde-carbonyl group. For the purposes of our invention, we can use the residue of the streptomycin molecule which results from the elimination of the aldehyde-carbonyl group, or the residue of a streptomycin molecule which has been modified in a group or groups thereof other than the aldehydecarbonyl group, the aldehyde-carbonyl group having been eliminated from the last-mentioned residue.

We believe that when streptomycin reactswi-th cysteine at neutral pH, a thiazolidine ring compound of streptomycin is formed. We believe that this derivative is inactive because of the size and shape of the thiazolidine ring group.-

The structural formula of thiazole, CsHaNSJs The structural formula of meta-thiazolidine,

CsHvNS, is

(5)H2C CH2(2) (4)Hz( )NH 3 The structural formula of ortho-thiazolidine is V mmo ,NH 2

(4)H2C-CH2(3) 'When streptomycin is reacted with l-cysteine,

the reaction may be represented as follows:

H N112 Strep-CHO HSCCCOOH s \H (5) Hit CStrep(2) (4)CO0H](EJ[-NH 3) This compound may be designated as, 2 Strepi-carboxy-thiazolidine;v

It is well-known that Raney nickel has a desulfurizing-action; and that-it also acts as a reducing catalyst with hydrogen. This-is disclosed in Journal of American Chemical Society (1943), vol. 65, page 1013; :also VolIJGfi/(IQM),

3 page 909; also vol. 68 (1946'), pages 724 and 1455. Reference is also made to Journal of Biological Chemistry, vol. 146, page 47-5. Raney nickel is nickel hydride or a composition which contains nickel hydride.

In using Raney nickel, the use of a hydrogen atmosphere is optional. As usually prepared, Raney nickel contains sufficient adsorbed hydrogram-positive gen to permit it to produce simultaneous desul furizing and hydrogenating effects. M

Our invention is further disclosed in the following description and examples, which disclose several examples of the "class which we designateas amino-mercapto compounds.

EXAMPLE NO. l

131.5 milligrams of partially purified streptO mycin constituting a total of 80,951 units" or micrograms of streptomycin base, were'di's'solve'd that 21.5% of the original streptomycin antibiotic activity was still present. 30 additional milligrams of cysteine hydrochloride were dissolved in the water of saidsolution, and the pH was adjusted-to 7.0. The solution was kept at 375 C. for an additional period of 3 hours. was less than 1.9% residual antibiotic activity left after this treatment, as'compared with the starting material.

This solution was mixed with water, up to a volume of 25 cubic centimeters. 1.25 grams of Raney nickel were added to the solution, and themixture was agitated at room temperature of C. C. for 6 hours, in a hydrogen atmosphere which was at a pressure of 760 mm.

of mercury. 7 v

The final pH was 6.5. The solution, after filtration, assayed 47,381 plus or minus 1661 units (calculated to streptomycin). This constituted about 55% of the original activity, after correcting for samples taken for assay. The new product, in'crude form, may be isolated; by evaporating the solution to a small volume, dissolving the new product in methanol by mixing said concentrated solution with anhydrous methanol, and precipitating the new product from the-methanol solution with several volumes of ethyl ether.

Some of the product'is adsorbed by Raney nickel. Since, in treating a solution of the new product with fresh Ra'ney nickel, there was a diminution in antibiotic activity, of the new product, this suggests that the new product which is formed by treating the streptomycincy'steine "re'actionprodu'ct with Raney nickel, may have at least and possibly morethan 55% of the activity of streptomycin itself on a mol for mol basis. Y i a From the method of synthesis employed, the new active derivative may be represented as,

CHa

H H StrepC-NCC O OH H H If the group,

H Strep-C- alanine, since the formula of 1-'ai1anil1flS There In treating this new compound with cysteine in the manner above described, no inactivation of the new compound results. This shows the absence of a free aldehyde group in this new compound. The antibiotic referred to as N streptomycyll-alanine is active as an antibiotic against many and gram-negative micro-0rganisms.

These micro-organisms include B. subtilzs, B. myooides, S. aureus 209 P., E. Coli, S. maroescens, and unclassified gram-negative coeco-bacillus.

EXAMPLE NO. 2

In this example, we used alpha-mercaptoethylamine to react with streptomycin, in order to produce a streptomycin derivative of low antibiotic activity.

The formula of alpha-mercapto-ethylamine is I-ISCHaCI-IaNHz We have used alpha-mercapto-ethy1amine hydrochloride, namely,-

' nscmomivmnoi Since the mercapto group and the amino group are attached to adjacent carbon atoms, each of said compounds may be designated, for the purposes of our invention,- as an alpha-aminomercapto compound.

Upon treating this substantially inactive derivative of streptomycin with Raney nickel, a large regeneration of antibiotic activity was obtained.

A mixture of 211 milligrams of partly purified streptomycin (representing 109,000 plus or minus 9080 units or micrograms of streptomycin base), and 113 milligrams of beta-mercaptoethylamine hydrochloride was dissolved in 10 cubic centimeters of water; the pH was adjusted to "7.0 with an aqueous solution of sodium hydroxide, and the resulting solution was heated at 50 C. for minutes. Assay at the end of this period gavea recovery of 17,500 plus orminus 770 units, which is equivalent to 16% of the original activity. Therefore, 84% of -the original streptomycin activity had disappeared.

A mixture of 5 cubic centimeters of the reaction mixture, 10 cubie centimeters of water, and 0.4 gram of Ran'ey nickel catalyst was shaken under atmospheric pressureof 760mm. of mercury in an atmosphere of hydrogen, .and at room temperature'of 20f C'.-25 C., for 4.5 hours. The solution was filtered. I

Bacterial assay by the usual cup plate-method and: extrapolation to the total quantity of starting material gave a recovery of 67,800; plus or minus 3850 streptomycin units, or 62% calculated tostreptomycin.

. If the, 16% activity remainingbefore desulfurization and reduction with Raney nickel is subtractedfrom the 62%, a 46% net increase in antibiotic activity'of the original derivative has been obtained. I v r From-thesteps employed in the synthesisit is highly probable that we have prepared what may be called N streptomycylethylamih.

'In; another example of our method; streptomycin-was substantially inactivated by alphamercaptoethylamine in anhydrous methanol. On'treating with Raney nickel in said methanol solution, an increase in activity similar to that noted above was observed. This shows that the ,formation of, an active streptomycin derivative -irom the substantially inactive reaction product which is; obtained when streptomycin and betamercaptoethylamine are reacted, can occur-in a non-aqueous medium under the; influence of Raney nickel. 1

EXAMPLE NO. 3.

A. Water as solvent A mixture of 212 milligrams of partially puri-' fied streptomycin (representing 145,000 plus or minus 3820 units or micrograms of streptomycin base) and 204 milligrams of l-cysteine ethyl ester hydrochloride, was dissolved in 10 cubic centimeters of water at 20 C.-25 C. The pH of this aqueous solution was adjusted to 6.9 with an aqueous solution of alkali, and the resultant solution, having a volume of 10.6 cubic centimeters, was heated at 50 C. for 1.5 hours, thus producing a solution of the substantially inactive derivative of streptomycin.

Bacterial assay at the end of this period indicated that 4130 plus or minus 387 units remained in said derivative. Therefore, this represented a 97% inactivation.

A mixture of 5 cubic centimeters of said solution, cubic centimeters of water, and 0.75 gram of Raney nickel catalyst was shaken in an atmosphere of hydrogen at 25 C. for 4.5 hours, under a pressure of 760 mm. of mercury. Bacterial assay using S. aureus 209 P, which is a strain of S. aureus, and extrapolation to the total amount of starting material, gave a recovery of 36,600 plus or minus 6480 units calculated to streptomycin or 25% recovery.

B. Anhydrous methanol as solvent A mixture of 214 milligrams of partly purified streptomycin (representing 128,000 plus or minus 9200 units or micrograms of pure streptomycin base), and 221 milligrams of l-cysteine ethyl ester, was dissolved in 9 cubic centimeters of anhydrous methanol at C.- C., and this solution was mixed with 0.4 cc. of a 1.85 M solution of sodium methylate, CH3ONa, in anhydrous methanol. The resulting clear solution was heated at 50 C. for 1 hour, to produce the solution of the inactive derivative. Bacterial assay at the end of this time showed a recovery of 7980 plus or minus 1440 units, which corresponds to an inactivation of 94%.

A mixture of 5 cubic centimeters of said solution, 10 cubic centimeters of anhydrous methanol,

and 0.75 gram of Raney nickel catalyst wasshaken in an atmosphere of hydrogen, at 25 C. for 615 hours, at a pressure of 760 mm. of mercury.

Bacterial assay and extrapolation to the total amount of starting material, gave a recovery of 49,600 plus or minus 6530 units calculated as streptomycin, or a 39% recovery.

The new derivative formed in either method of this example, is probably the ethyl ester of N-streptomycyl-l-alanine.

It has been found that the amount of Raney nickel used is important. If too much is used, the products described in the examples above may be lost on the Raney nickel. If too little Raney nickel is used, the Raney nickel is apparently used up by the excess of mercapto reagent used, and no activity will be recovered. On adding more Raney nickel the activity can then-be recovered. This wasespecially true for the treatment with Raney nickel of the reaction product of streptomycin and l-cysteine, .as'in ExampleNo.1...: Y I .Forany given case it is a simple matter-to 'dee termine by trial the best amount of Raney nickel to use.

While in the above examples, an atmosphere of hydrogen was provided during the hydro-' genolysis, We have found that the use of aliydrogen atmosphere is not necessary because .the Raney nickel itself provides enough adsorbedhydrogen for the desired reaction. x

.We can reactstreptomycin with any one ofaa number of, reagents which contain the. alpha? amino mercapto groups, and the resulting.inactive derivative can then be treated. with Raney nickel togive an active antibiotic.

Instead of using streptomycin as. a starting material to. react with cysteine or its substituents, we. can use derivatives of streptomycin which have retained unmodified the streptose aldehydecarbonyl group, CHO. 1 Instead of using l-cysteine, we can use d-cysteine or the racemic form, namely dl-cysteine.

Instead of using cysteine,:we can use peptides whichcontain cysteine, either 1 or d or dl. Such peptides are exemplified by cysteinyl-glycine.

Cysteine is an example. of mercapto amino acids, and we can use such acids generally, and their acyl and aryl esters.

We can also use dipeptides. or higher dipeptides which include these mercapto amino acids.

We can also use substituted cysteine residues, either 1 or d or racemic, such as beta-beta-dimethyl cysteine, or beta-methyl-cysteine.

In general, we can also react streptomycin or its derivatives which contain the streptose aldehydic carbonyl group, with aliphatic or aromatic residues represented by,

H H ,H t t R IlTHz SIH wherein R is a straight chain alkyl or aryl substituted straight chain alkyl group..

Additionally, we can react streptomycin or one of its functional derivatives containing the streptose :aldehydic carbonyl group, with a substance represented by the formula, in which R1, R2, R3, and R4 individually have the meaning above assigned to R, as follows:

The resultant reaction compound is then treated with Raney nickel to desulfurize said reaction compound, with simultaneous reduction of said resultant reaction compound.

We have described numerous embodiments of our invention, but numerous changes and omissions and additions can be made without departing from its scope.

Thus, We generally claim antibiotic compounds of streptomycin and amino-acids, particularly with amino-acids which occur in proteins, in which the designation streptomycin includes derivatives of streptomycin which are reactive with sulfur-containing amino-acids, particularly those which occur in proteins. amino-acids which have been isolated from proteins, as stated at pages 418-419 of Organic Chemistry, by Desha, published in 1936 by McGraw-Hill Book Company Inc., include glycine,

The principal V serine; phenylalanine;tyrosine, cystine, proiine, and'ihydroxyproline. a .A'sztabove: stated, and according to .oneephase. of our invention, we finally produce anopenachain sulfur-free. compound, in; which the chain; consistsof carbon and nitrogen atoms.

We also generally claim substituted streptomyclamine derivatives of streptomycin as antibi'oticfs, whereat least'one hydrogen .atom of theiNHz' group of streptomyclamine isyreplaced bysa substituent.

' We-claim:

1. The process for manufacturing aniantibiotic agent-related to'streptomycinthat comprises reacting: aos'ubstance ofthe group consisting of streptomycin and its derivatives having the characteri'stic streptose aldehyde-carbonyl group, with an alpha aminoi mercapto straight chain aliphatic compoundin a solvent reaction'medium, then; reducing this reaction product with Raney nickel to' produce anantibiotically active substance. idifiering from the starting material in that the streptose carbonyl group is replacedrby a 'substituent of the class consisting of alkyl amino'm'ethylene groups and: substituted alkyl aminoi methylene groups of the lower alkyl series, and recovering this hydrogenated compound from the-reaction mixture. t

A process as defined in claim 1 further characterized' in that theihydrogenation product'recovered is a compound of the group-consisting 'of N-(l -carboxy) -ethyl streptomycylamine, N-

8 ethyl streptomycylamineiand Nail-carboeithoxy) ethyl streptomycylamine. I j r 3. As an antibiotically active streptomycimderivative, an N-monc-substituted streptomycylamine wherein the substituent group is of the class consisting-pf lower straight chain -alkyl groups and :aryl substituted lower straight chain alkylgroups; l '2 v 4. An anti'biotically active streptomycin 'de-' rivative-as defined in claim 3whereinthe-substituent group is a lower straight-chain alkyl group.

5. Anantibiotically active streptomycin derivative as definedin claim 3 whereinthe substituentgroup is an ethy-lgroupn I p 6. An= antibiotically' active streptomycin derivative as defined inclaim 3 wherein the sub* stituent group is-an 1%carboxy-ethyl group.

7. An antibiotically active streptomycin derivative as-defin-ed in" claim 3 wherein theu'substituent group is-an' l-carboethoxy-ethyl group;

WALTER A. WII TSTFI I; MURRAYKA'ITZ. SIDNEY R; SAFIR. Y

REFERENCES CITED The following references are-of record; in the file of'this patent:

'Denkelwa-ter et'alv: Science, v. 102 (1945), page 12. 

1. THE PROCESS FOR MANUFACTURING AN ANTIBIOTIC AGENT RELATED TO STREPTOMYCIN THAT COMPRISES REACTING A SUBSTANCE OF THE GROUP CONSISTING OF STREPTOMYCIN AND ITS DERIVATIVES HAVING THE CHARACTERISTIC STREPTOSE ALDEHYDE-CARBONYL GROUP, WITH AN ALPHA AMINO MERCAPTO STRAIGHT CHAIN ALIPHATIC COMPOUND IN A SOLVENT REACTION MEDIUM, THEN REDUCING THIS REACTION PRODUCT WITH RANEY NICKEL TO PRODUCE AN ANTIBIOTICALLY ACTIVE SUBSTANCE DIFFERING FROM THE STARTING MATERIAL IN THAT THE STREPTOSE CARBONYL GROUP IS REPLACED BY A SUBSTITUENT OF THE CLASS CONSISTING OF ALKYL AMINO METHYLENE GROUPS AND SUBSTITUTED ALKYL AMINO METHYLENE GROUPS OF THE LOWER ALKYL SERIES, AND RECOVERING THIS HYDROGENATED COMPOUND FROM THE REACTION MIXTURE. 